Induction and persistence of cytogenetic damage in mouse splenocytes following whole-body X-irradiation analysed by fluorescence in situ hybridization. II. Micronuclei.

Micronuclei are formed during cell division either from lagging acentric fragments caused by chromosomal breakage or from whole chromosomes. Female Swiss mice were used to study the induction and persistence of micronuclei (MN) in isolated splenocytes up to 112 days after 2Gy whole-body X-irradiation. Micronucleus frequency in cytokinesis blocked binucleated cells was estimated using acridine orange staining. Fluorescence in situ hybridization (FISH) with murine minor satellite DNA probe was used to detect aneuploidy (MN with a centromere). The initial frequency of micronuclei immediately after X-ray exposure was found to be 39.6 per 100 cells. The MN frequency declined in an exponential manner during the subsequent period and at day 14, reached about half the value observed immediately after irradiation with a further 50% reduction by day 28 and only 3% MN could be detected at day 112 post-irradiation. Some induction of aneuploidy was observed after X-irradiation, deduced in the form of centromere-positive micronuclei (C + MN) as detected by the presence of a minor satellite signal after FISH. Of MN, 23% were centromere-positive in the first fixation time after X-ray exposure. Some of the MN showed two or more centromeric signals. The percent C + MN increased gradually until day 7 post-exposure. At day 14, this value started to decline, reaching 13% by 112 days post-irradiation. FISH with a biotinylated minor satellite probe can reliably be used in the cytokinesis blocked micronucleus assay to distinguish the clastogenic and/or aneugenic activity of a test chemical or physical agent.